Separation of triterpenoid saponins from the root of Bupleurum falcatum by counter current chromatography: The relationship between the partition coefficients and solvent system composition

A separation method using counter current chromatography coupled with an evaporative light‐scattering detection system was developed to purify five triterpenoid saponins from the roots of Bupleurum falcatum. The methanol extract was loaded onto a Diaion® HP20 column and fractionated by a methanol and water gradient elution. The saikosaponin‐enriched fraction was obtained by elution with 100% methanol. The two‐phase solvent systems used for separation were composed of chloroform/methanol/isopropanol/water at a volume ratio of 60:60:1:60 and 6:6:1:6. The relationship between the isopropanol ratio of each phase and the partition coefficients of the target compounds was investigated by calculating partition coefficient by high‐performance liquid chromatography and measuring the accurate composition of each phase by ¹H NMR spectroscopy. Each fraction obtained was collected and dried, which yielded the following five saikosaponins from 700 mg of injected sample: saikosaponin B₁ (8.7 mg), saikosaponin A (86 mg), saikosaponin B₃ (17 mg), saikosaponin B₂ (41 mg), and saikosaponin C (33 mg). Saikosaponin A showed the most potent cytotoxicity against human cancer cells (gastric cancer, AGS cells; breast cancer, MCF‐7 cells; and hepatoma, HepG2 cells) after 24 h. The IC₅₀ values for the above three cell types were 34.6, 33.3, and 23.4 μmol/L, respectively.     

Combination of supercritical fluid extraction with counter‐current chromatography to isolate anthocyanidins from the petals of Chaenomeles sinensis based on mathematical calculations

Supercritical fluid extraction (SFE) coupled with high‐speed counter‐current chromatography (HSCCC) was successfully used for the extraction and on‐line isolation of the anthocyanidins from the petals of Chaenomeles sinensis in two stages. The SFE parameters were optimized by an orthogonal test, and the solvent systems of SFE and HSCCC were calculated and optimized with the help of a multiexponential function model. In the first stage, the lower phase of the solvent system of n‐butanol/tert‐butyl methyl ether/acetonitrile/0.1% aqueous TFA (0.715:1.0:0.134:1.592, v/v/v/v) was used as both the SFE modifier and the HSCCC stationary phase, after extraction, the extractants were pumped into HSCCC column, and then eluted with the corresponding upper phase to isolate the moderately hydrophobic compounds. In the second stage, the upper phase of the solvent system of n‐butanol/ethyl acetate/acetonitrile/0.1% aqueous TFA (1.348:1.0:0.605:2.156, v/v/v/v) was used as both the SFE modifier and the HSCCC stationary phase, followed by elution with the corresponding lower phase to separate the hydrophobic compounds. With the help of two‐stage SFE/HSCCC, six compounds including delphinidin‐3‐O‐glucoside (Dp3G), cyanidin‐3‐O‐glucoside (Cy3G), peonidin‐3‐O‐glucoside (Pn3G), delphinidin (Dp), peonidin (Pn), and malvidin (Mv) were successfully separated within 300 min. The targeted compounds were identified by UV spectrophotometry, MS, and NMR spectroscopy. This research has opened up great prospects for the industrial application of SFE–HSCCC for the automatic extraction and separation of unstable compounds.